Novel lipids for transfection of nucleic acids

ABSTRACT

Cationic lipid compositions are provided that are useful for efficient delivery of macromolecules, such as nucleic acids, into a wide variety of eukaryotic cell types. Methods for using the compositions also are provided.

This application is a divisional application of U.S. patent applicationSer. No. 10/851,658, filed Mar. 25, 2004, which claims the benefit ofpriority under 35 U.S.C. §119(e) to U.S. Provisional Patent ApplicationSer. No. 60/472,403, filed May 22, 2003, the entireties of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

The use of cationic lipids to deliver nucleic acids into cultured cellswas first described by Felgner and co-workers (Proc. Nat'l Acad. Sci.84,7413 (1987)). Subsequently, Behr (Proc. Nat'l Acad. Sci. 86.6982(1989)) showed that polycationic lipids also can be effective deliveryagents. Large number of cationic lipid reagents have now been describedand several of these reagents are commercially available, for example,LipofectAmine, LipofectAmine 2000, Fugene, TransfectAm, Lipofectin andDOTAP. None of these reagents, however, is universally effective on allcell lines and none is as effective as viral based gene deliverysystems. In addition, most of the reagents are toxic in some degree tothe cells being transfected.

Accordingly, there still exists a great need for the development oftransfection reagents that are less toxic than those currently availablebut that are highly efficient and more universally applicable fortransfecting a wide variety of cell types. Ideally, such a reagent willhave minimal safety risk and immunogenicity when compared to viral baseddelivery systems. The successful development of such a reagent will havea profound impact in biotechnology in general and gene therapy inparticular.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide novel compositionsthat are useful for delivery of macromolecules, such as nucleic acids,into cells.

It is a further object of the invention to provide methods of usingthese compositions to deliver macromolecules, such as nucleic acids,into cells.

In accomplishing these objects, there is provided, in accordance with afirst aspect of the invention, a lipid having the formula:

wherein X is N(R⁴R⁵R⁶) or dialkylphosphatidyl, or X is

Y and Z independently are selected from the group consisting of alkoxy,alkanoyloxy, alkylamine, alkyl urethane and alkyl guanidine

R¹ and R² independently are selected from the group consisting ofhydrogen, C₁-C₆ alkyl, alkylamine, alkylaminoalcohol, spermiyl,spermidyl and carboxyspermiyl,

R³ is H or C₁-C₄ alkyl,

R⁴, R⁵, and R⁶ independently are selected from the group consisting ofhydrogen, alkyl, alkenyl, aryl and alkylaryl, provided that at least oneof R⁴, R⁵, and R⁶ is a long chain alkyl or alkenyl,

W is short chain alkyl or alkylamino, and

R⁷ is a negative charge or short chain alkyl.

In accordance with a second aspect of the invention there is provided alipid having the formula:

wherein X¹ is selected from the group consisting of (CH₂)_(n),(CHOH)_(n), —NH—, —O—, a polyether, an ester linkage —S—, CONH, NHCO, apolyamide —NHCONH—, and NHC(═NH) NH,

wherein Y′ is selected from the group consisting of (CH₂)_(n), apolyether, NHCO, —CO—, and a polyamide,

wherein Y² is selected from the group consisting of (CH₂)_(n), —NH—,—O—, a polyether, an ester linkage, —S—, CONH, NHCO, a polyamide—NHCONH—, and NHC(═NH)NH,

wherein Z¹ and Z² are independently selected from the group consistingof hydrogen, primary alkylamine, secondary alkylamine, tertiary alkylamine, quaternary alkylamine, alkyl amino alcohol, alkyl polyamine,spermidine, spermine, carboxy spermine, guanidinium, pyridinium,pyrollidinium, piperidinyium amino acyl, peptidyl, and protein,

R⁸, R⁹, R¹⁰, and R¹¹ are independently absent or are selected from thegroup consisting of hydrogen, straight chain C₁-C₂₀ alkyl, branchedalkyl, cyclalkyl, straight chain alkenyl, branched alkenyl, cycloalkenyl, and optionally substituted aryl, wherein said optionalsubstitution, when present, comprises at least one functional groupselected from the group consisting of —OH, NH₂, —COOH, ester, amide,alkylamino, —NHCONH—, and NHC(═NH) NH], and

wherein n=1-12.

In one embodiment, there is provided a composition comprising a lipid asdescribed above, and one or more macrolecules, such as one or morepeptides, proteins and/or nucleic acids. The nucleic acid may comprise aDNA molecule, for example a double stranded DNA molecule. The DNAmolecule may be a plasmid, and may encode, for example, an RNA moleculethat is self complementary and that forms a region of double strandedRNA.

In another embodiment, the nucleic acid may comprise one or more RNAmolecules, for example, one or more double stranded RNA molecules. TheRNA molecule may be an siRNA.

In accordance with another aspect of the invention there is provided amethod of introducing a peptide, protein, and/or nucleic acid into acell or a tissue, by contacting a eukaryotic cell or tissue with acomposition as described above.

In accordance with yet another aspect of the invention there is provideda kit for transfecting a cell, comprising a lipid as described above.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results obtained from transfection of COS 7 cells withpCMV-Sport β-gal and MT-R1, MT-R2, MT-R3 and LipofectAMINE 2000 (L2K).

FIG. 2 shows the results obtained from transfection of VERO cells withpCMV-Sport β-gal using MT-R1 and commercial cationic lipids.

FIG. 3 shows the results obtained from transfection of BHK-21 cells withpMCMV-Sport β-gal MT-R2 and lipofectamine 2000.

FIG. 4 shows a generalized synthesis of cationic glycolipid analogs

FIG. 5 shows a synthesis of alkylaminoalcohol lipids

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel lipids that are effective for theintroduction of nucleic acids and peptides and proteins into a widevariety of cells and tissues. The lipids are highly efficient anddemonstrate low toxicity in all cell types tested. The lipids have thegeneral structures I and II shown below.

The compounds of formula I contain an amino alcohol head group, and Xmay be N(R⁴R⁵R⁶) or dialkylphosphatidyl, or X may be

Y and Z independently may be selected from the group consisting ofalkoxy, alkanoyloxy, alkylamine, alkyl urethane and alkyl guanidine. R¹and R² independently may be selected from the group consisting ofhydrogen, C₁-C₆ alkyl, C₁-C₆alkylamine, alkylaminoalcohol, spermiyl,spermidyl and carboxyspermiyl. R³ is H or C₁-C₄ alkyl. R⁴, R⁵, and R⁶independently are selected from the group consisting of hydrogen, alkyl,alkenyl, aryl, and alkylaryl, provided that at least one of R⁴, R⁵, andR⁶ is a long chain alkyl or alkenyl. The alkyl, alkenyl, aryl, andalkylaryl groups may contain, for example, 6 to 30 carbon atoms,advantageously 10 to 18 carbon atoms, although the skilled artisan willrecognize that the groups may contain fewer than 6 or more than 30carbon atoms. W may be short chain (C₁-C₆) alkyl or alkylamino, and R²may be a negative charge or short chain (C₁-C₆) alkyl. Representativeexamples of compounds encompassed by general formula I include compounds12 and 13 shown below, where for example, R is oleyl.

In compounds of formula II, which contain hydrocarbon moietiesconjugated to simple carbohydrates, X′ may be selected from the groupconsisting of (CH₂)_(n), (CHOH)_(n), —NH—, —O—, a polyether, an esterlinkage —S—, CONH, NHCO, a polyamide —NHCONH—, and NHC(═NH) NH. Y¹ maybe selected from the group consisting of (CH₂)_(n), a polyether, NHCO,—CO—, and a polyamide. Y² may be selected from the group consisting of(CH₂)_(n), —NH—, —O—, a polyether, an ester linkage, —S—, CONH, NHCO, apolyamide —NHCONH—, and NHC(═NH)NH. Z¹ and Z² may independently beselected from the group consisting of primary alkylamine, secondaryalkylamine, tertiary alkyl amine, quaternary alkylamine, alkyl aminoalcohol, alkyl polyamine, spermidine, spermine, carboxy spermine,guanidinium, pyridinium, pyrollidinium, piperidinyium amino acyl,peptidyl, and protein. R⁸, R⁹, R¹⁸, and R¹¹ independently may be absentor may be selected from the group consisting of hydrogen, straight chainC₁-C₂₀ alkyl, branched alkyl, cyclalkyl, straight chain alkenyl,branched alkenyl, cyclo alkenyl, and optionally substituted aryl, wherethe optional substitution, when present, comprises at least onefunctional group selected from the group consisting of OH, NH₂, —COON,ester, amide, alkylamino, —NHCONH—, and NHC(═NH)NH], and n is an integerfrom 1 to 12.

In the context of the present invention, a short chain alkyl group istypically, unless otherwise defined, C₁-C₆ alkyl, for example. A longchain alkyl group is typically, unless otherwise defined, C₁₀-C₂₀ alkyl,for example, or C₁₀-C₃₀ alkyl. When not specifically defined, eitherdefinition may be used, as appropriate. The skilled artisan also willappreciate that other derivative groups containing alkyl moieties, forexample, alkoxy moieties and the like, also may contain short and/orlong chain groups as appropriate in the context, unless otherwisedefined.

Molecules of formulas I and II may be prepared by methods that are wellknown in the art. See, for example, U.S. Pat. Nos. 5,334,761 and5,264,618, WO00/27795 and Benerjee et al. (J. Med. Chem., 44.4176(2001), which references are hereby incorporated by reference in theirentireties.

Specific methods are shown below for the synthesis of representativemembers of both classes of lipids (see Scheme 1 and Scheme 2 in FIGS. 4and 5). The skilled artisan will recognize that other members of theselipid classes can be synthesized using variations of these methods or byother methods that are well known in the art.

Preparation of the Lipids

Synthesis of the cationic glycolipids (II) may be achieved via themethods outlined in Scheme 1 (FIG. 4). 2,3-Isopropylidine tartaricanhydride is treated with an alkylamine, such as oleoyl amine, toring-open the anhydride and generate the corresponding amide andcarboxylic acid. Treatment of the amide with acidic methanol results inthe removal of the isopropylidine group and esterification of the acid.The ester is treated with alkylamine such as methylamine to obtain thecorresponding diamide. The diamide is reduced to the diamine with areducing agent such as lithium aluminum hydride and alkylated withalkylating agents such as bromopropylphthalimide. The phthalimide groupis removed with hydrazine and the amine protonated with HCl to obtainthe desired cationic lipid. This scheme can be effectively used tosynthesize specific compounds of the general structure1-[(3-aminopropyl)-alkyl¹amino]-4-[(3-aminopropyl)-alkyl²-amino]-butane-2,3-diolwhen bromopropylphthalamide is used as the alkylating agent or1-[(3-amino-2-hydroxypropyl)-alkyl¹amino]-4-[(3-amino-2-hydroxypropyl)-alkyl²-amino]-butane-2,3-diol.Alkyl¹ can be methyl, ethyl, propyl, C₁₀-C₂₀ alkyl chain and alkyl² canbe a C₁₀-C₂₀ alkyl chain.

Alternatively, the symmetrical analogs of the cationic glycolipids canbe obtained starting with the amidation of the methyl ester of a desireddiacid (HO₂C—(CHOH)_(n)—CO₂H, n=2-6) such as dimethyltartarate with adesired alkyl amine. The diamide is reduced using a suitable reducingagent, such as lithium aluminum hydride, to generate the correspondingamine. The amine groups may be alkylated with bromopropyl phthalimide orepoxypropyl phthalimide, followed by removal of the phthalimide groupwith hydrazine hydrate to obtain the desired cationic lipid. Thissynthetic route is generally applicable to all compounds of generalformula 1,4-bis[(3-amino-propyl)aklylamino]-butane-2,3-diol whenbromopropyl phthalimide is used as the alkylating agent, and1,4-bis[(3-amino-2-hydroxypropyl)alkylamino]-butane-2,3-diol whenepoxypropylphthalamide is used as the alkylating agent. The alkyl groupcan constitute a C₁₀-C₂₀ alkyl chain. Various glycolipids can obtainedby starting with other alderic acids HO₂C—(CHOH)_(n)—CO₂H where n=2-6.Representative compounds may be formulated into liposome alone or withco-lipids (DOPE or cholesterole) and used in transfection.

Lipids of formula I having an amino alcohol head group may besynthesized as shown in Scheme 2 (FIG. 5). Dimethylaminopropane diol canbe alkylated with a desired alkyl sulfonate to producedialkyloxy-dimethyaminopropane. Further alkylation ofdialkyloxy-dimethylamnopropanc, to quaternize the amine group, withepoxypropyl phthalimide and the removal of the phthalimide protectinggroup with hydrazine hydrate provides the desired aminoalcohol as themajor product, together with a minor product derived from alkylation ofthe hydroxide group. Both compounds were protonated with HCl andformulated into liposome alone or with co-lipid (cholesterol or DOPE)and used in transfection.

Cell Transfections Using the Lipids.

The novel lipids described herein may be formulated with one or morenucleic acids into liposomes or liposome-like vehicles in the presenceor absence of co-lipid such as dioleylphosphatidyl ethanolamine (DOPE)or cholesterol. The lipids may be formulated into liposomes by themethod of reverse evaporation, which is well known in the art.Alternatively the lipids may be formulated by other well known methodsfor liposome formation such as sonication, microfluidization etc. Theseliposome formulations are effective for transfecting DNA into culturedcells.

The lipids are at least as active, and in most cases more active, thancationic lipids that currently are commercially available. The nucleicacid can be any type of nucleic acid that is known, provided that thenucleic acid is sufficiently negatively charged to form a lipidaggregate, liposome, or liposome-like complex when admixed with thelipid. The nucleic acid can be, for example, DNA or RNA, and can be anoligonucleotide, plasmid, or other form of nucleic acid molecule. Thenucleic acid may be, without limitation, an antisense molecule, or maybe a double stranded RNA molecule of the type used for inhibiting geneexpression by RNA interference. The nucleic acid may be a shortinterfering double stranded RNA molecule (siRNA).

The lipids may also be used to introduce peptides and proteins and thelike into cells using methods that are known in the art. Methods ofusing cationic lipids for peptide and protein delivery previously havebeen described.

In addition, the lipids may be used to deliver nucleic acids, peptidesand proteins and the like into tissues in vivo. Methods of using lipidsfor delivering compounds to tissue in vivo previously have beendescribed.

Liposome formulations derived from the above compounds were evaluatedfor transfection efficiency of cultured cells such as BHK-21, HeLa,COS-7, CHO-K1, VERO and 293 with β-galactosidase reporter plasmidpCMVe•SPORT-β-gal as described below.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

Example 1 Synthesis ofN-1-dimethyl-N-1-(2,3-ditetradecyloxypropyl)-2-hydroxypropane-1,3-diamine(13)

3-(Dimethylamino)-1,2-Propanediol (10.19 g, 85 mmole) was treated with2.5 equivalents of sodium hydride in tetrahydrofuran for a 18-24 hourperiod at 70°±475° C. After 18-24 hour, 3 equivalents oftetradecylmethane sulfonate (75.0 g, 256 mmole) was added to thereaction and allowed to reflux (70°±5° C.) for an additional 40-48hours. The reaction mixture was concentrated to dryness and subjected toextraction using dichloromethane and water. The organic layer was driedover sodium sulfate and concentrated to an oil on a rotary evaporator.The resulting residue was loaded on a normal phase flash chromatographycolumn and the desired product,1-dimethylamino-2,3-ditetradecyloxypropane, was purified using ethylacetate/hexane as the eluant. The final product was analyzed by TLC.

1-Dimethylamino-2,3-ditetradecyloxypropane (15.0 g, 29.35 mmole) wastreated with two equivalents of N-(2,3-epoxypropyl)-phthalimide (11.93g, 58.7 mmoles) and two equivalents of lithium perchlorate (6.245 g,58.7 mmole) using ethanol as a solvent. The reaction was performed at75-80° C. for a 96±6 hour period. The reaction was monitored forcompleteness and formation of desired products1(N,N-dimethyl-N(2-hydroxy-3-phthalimido-propyl)-ammonium-2,3,-ditetradecyloxypropane.The reaction mixture was concentrated to dryness and extracted usingdichloromethane and water. The organic layer was dried over sodiumsulfate and concentrated on a rotary evaporator. The resulting reactionmix was loaded on a normal phase flash chromatography column and thedesired product1-(N,N-dimethyl-N-(2-hydroxy-3-phthalimido-propyl)-ammonium-2,3,-ditetradecyloxypropanewas purified along with the O-alkylated byproduct usingchloroform/methanol as the eluant. The mixture (14.95 g) was reactedwith hydrazine hydrate (4.6 ml) using ethanol as a solvent at 75-80° C.for 15-18 hours. The reaction was placed at 4° C. for an overnightperiod and the precipitate was filtered off. The filtrate was subjectedto rotary evaporation which resulted in a gummy material composed of amixture of two compounds. The residue was dissolved in tetrahydrofuranand acidified using 1.1 equivalents of hydrochloride acid for 1 hour atroom temperature. The reaction was dried down and co-evaporated twicewith methanol and twice with dichloromethane. The two compounds werepurified by reverse phase (C-18) flash chromatography usingmethanol/water as the solvent. The compounds were characterized by TLCand mass spectrometry asN-1-dimethyl-N-1-(2,3-ditetradecyloxypropyl)-2-hydroxypropane-1,3-diamine(major) andN-1-dimethyl-N-1-(2,3-ditetradecyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)-propane-1,3-diamine(minor)

Example 2 Synthesis of1-[(3-amino-2-hydroxypropyl)-alkylamino]-4-[(3-amino-2-hydroxypropyl)-alkylamino]-butane-2,3-diolor 1,4-Bis[(3-amino-2-hydroxypropyl)-alkylamino]-butane-2,3-diol

Dimethyl tartrate (8.2 g, 46.6 mmoles) was dissolved in 150 ml methanoland 1-alkylamine (0.11 mole) was added. The solution was heated toreflux overnight (>22 hrs). The reaction mix was cooled to roomtemperature and kept at 4° C. for about 1 hour. The precipitate wasfiltered, and washed twice with 50 ml of cold methanol (<4° C.) toobtain dialkyltartaramide as a white crystalline material (>80%). Theproduct was characterized by TLC and mass spectrometry.

The dialkyltartaramide (29.6 mmole) was suspended in 500 ml anhydrousTHF and 200 ml of 1 M lithium aluminium hydride solution in THF wasadded drop-wise. After the addition was completed, the reaction mix wasrefluxed overnight. The mixture was cooled and diluted with 500 ml THF.200 ml of 15% NaOH solution was added drop-wise to the mixture andstirred overnight. The THF layer was decanted and the remainingsuspension was exhaustively extracted with chloroform using TLC tomonitor the presence of the desired product in the chloroform layer. Theorganic layer was combined to obtain compound1,4-bis(alkyl-amino)-butane-2,3-diol' as a semi-solid (40-75% yield).The product was characterized by mass spectrometry.

1,4-Bis(alkyl-amino)-butane-2,3-diol (21.87 mmoles) was treated withN-(2,3-epoxypropyl)-phthalimide (10.90 g, 53.69 mmole) and lithiumperchlorate (5.63 g, 53.6 mmole) in 500 ml 10% reagent alcohol. Themixture was refluxed overnight and the reaction volume was reduced to100 ml using the rotary evaporator. The reaction mix was cooled anddiluted with 500 ml chloroform. The chloroform solution was extractedtwice with 300 ml sodium bicarbonate solution (0.1 M) and once with 300ml concentrated NaCl solution. The chloroform was removed on the rotaryevaporator to obtain the bis-phthalimide adduct as a gum. The compoundwas characterized by mass spectrometry.

Hydrazine hydrate (2 ml) was added to a solution of the phthalimide in100 ml of 100% reagent alcohol. The reaction mix was refluxed overnightand cooled to room temperature for about 1 hr. The reaction mix was thencooled at 4° C. overnight and the precipitate filtered off. The solidwas washed twice with 20 ml chilled ethanol (−20° C.). The ethanol wasremoved on the rotary evaporator. The solid was dissolved in chloroform,filtered and extracted with 200 ml water. The chloroform was removed onthe rotary evaporator to give a quantitative yield of the desiredcompound,1,4-bis[(3-amino-2-hydroxy-propyl)-alkyl-amino]-butane-2,3-diol. Thismaterial was purified on reverse phase (C-18) flash chromatography usingaqueous methanol as eluant and characterized by TLC and massspectrometry. In this manner compounds with alky groups varying inlength from C₁₂ to C₁₈ were synthesized.

Example 3 Formulation of Cationic Lipids into Liposomes

In general the required amount of the cationic lipid and the co-lipidare weighed and transferred into a round bottom flask. An amount ofchloroform that is enough to dissolve the lipids is added followed bysufficient molecular biology grade water to make the desiredconcentration of total lipids/volume (e.g. 2 mg/ml). The solution isplaced on the rotary evaporator and the chloroform removed under vacuum.As the chloroform is removed, liposomes are formed in the aqueousmedium. The solution becomes opalescent and varies in its turbiditydepending on the cationic lipid and co-lipid being formulated. Forexample 10.4 mg of1,4-bis[(3-amino-2-hydroxy-propyl)-tetradecyl-amino]-butane-2,3-diol and9.6 mg of DOPE (1:1 molar ratio) were combined and placed in a roundbottom flask. The lipid mixture was dissolved in 2 ml of chloroform. 10ml of water was added to the chloroform solution. The chloroform wasremoved under vacuum on the rotary evaporator to obtain a liposomesolution of 2 mg/ml (MT-R1). SimilarlyN-1-dimethyl-N-1-(2,3-ditetradecyloxypropyl)-2-hydroxypropane-1,3-diaminewas formulated with DOPE (1:1) (MT-R2) andN-1-dimethyl-N-1-(2,3-ditetradecyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)-propane-1,3-diaminewas formulated into a liposome by itself (MT-R3) as well as with DOPE.

Example 4 Transfection Protocol

Transfection of BHK-21, HeLa, COS-7, CHO-K1, VERO and 293 withβ-galactosidase reporter plasmid pCMV•SPORT-β-gal was carried out asfollows:

Cells were plated in a 96-well plates with 100 μl of media containing10% fetal calf serum the day before transfection such that a desiredconfluency (70%-95%) was achieved. The following day lipid and DNA weremixed in Opti-MEM to form DNA/lipid complexes. Complexes were formed byadding various amounts of lipids (0.1 to 1.0 μl) to 100 μl of Opti-MEM.DNA (50 ng to 400 ng) was added to 100 μl Opti-MEM. The DNA and lipidssolutions were then mixed to form DNA lipid complexes. The complexeswere incubated at least for 20 minutes and 20 μl of complexes were addeddirectly to the cells in 10% serum. Cells were incubated for anadditional 24 hours to allow expression of the plasmid. Medium wasremoved and the cells were lysed in 100-200 μl of lysis buffer. Thelysates (20 μl) were assayed for β-gal activity using the enzymaticsubstrate ONPG. Total activity was determined by reading the OD at 405using Bio-Rad Benchmark Microplate Spectrophotometer.

For siRNA transfection, a 24 well plate is seeded with the appropriatenumber of cells in serum containing medium a day before transfectionsuch that they will be 50 to 60% confluent and incubated at 37° C. in a3-5% CO₂ incubator overnight. For each well to be transfected 25 μl ofserum free medium containing 0.2 to 0.4 μl of lipid and 25 μl ofserum-free medium containing siRNA is prepared. Final concentration ofsiRNA is 10 nM. The lipid and siRNA solutions are mixed and incubated atroom temperature for 20 minutes. The lipid/siRNA complex (50 μl) isadded to the cells in serum containing medium and the cells areincubated at 37° C. in CO₂ incubator. Gene silencing can be monitored at24 to 72 hours after transfection.

Representative results are shown in FIGS. 1-3 below.

1. What is claimed is: A lipid having the formula I or II:

wherein in compounds of formula I: X is N(R⁴R⁵R⁶) ordialkylphosphatidyl, or X is

Y and Z independently are selected from the group consisting of alkoxy,alkanoyloxy, alkylamine, alkyl urethane and alkyl guanidine R¹ and R²independently are selected from the group consisting of hydrogen, C₁-C₆alkyl, alkylamine, alkylaminoalcohol, spermiyl, spermidyl andcarboxyspermiyl, R³ is H or C₁-C₄ alkyl, R⁴, R⁵, and R⁶ independentlyare selected from the group consisting of hydrogen, alkyl, alkenyl, aryland alkylaryl, provided that at least one of R⁴, R⁵, and R⁶ is a longchain alkyl or alkenyl, W is short chain alkyl or alkylamino, and R⁷ isa negative charge or short chain alkyl, and wherein in compounds offormula II, X¹ is selected from the group consisting of (CH₂)_(n),(CHOH)_(n), —NH—, —O—, a polyether, an ester linkage —S—, CONH, NHCO, apolyamide —NHCONH—, and NHC(═NH)NH, Y¹ is selected from the groupconsisting of (CH₂)_(n), a polyether, NHCO, —CO—, and a polyamide, Y² isselected from the group consisting of (CH₂)_(n), —NH—, —O—, a polyether,an ester linkage, —S—, CONH, NHCO, a polyamide —NHCONH—, and NHC(═NH)NH,Z¹ and Z² are independently selected from the group consisting ofhydrogen, primary alkylamine, secondary alkylamine, tertiary alkylamine, quaternary alkylamine, amino alcohol, alkyl polyamine,spermidine, spermine, carboxy spermine, guanidinium, pyridinium,pyrollidinium, piperidinyium amino acyl, peptidyl, and protein, R⁸, R⁹,R¹⁰, and R¹¹ are independently absent or selected from the groupconsisting of hydrogen, straight chain C₁-C₂₀ alkyl, branched alkyl,cyclalkyl, straight chain alkenyl, branched alkenyl, cyclo alkenyl, andoptionally substituted aryl, wherein said optional substitution, whenpresent, comprises at least one functional group selected from the groupconsisting of —OH, NH₂, —COOH, ester, amide, alkylamino, —NHCONH—, andNHC(═NH)NH], and n=1-12.
 2. The composition comprising a lipid accordingto claim 1 and a macromolecule.
 3. The composition according to claim 2wherein said macromolecule is a nucleic acid.
 4. The compositionaccording to claim 3, wherein said nucleic acid comprises a DNAmolecule.
 5. The composition according to claim 4, wherein said DNAmolecule is a double stranded DNA molecule.
 6. The composition accordingto claim 5, wherein said DNA molecule is a plasmid.
 7. The compositionaccording to claim 6, wherein said plasmid encodes an RNA molecule thatis self complementary and that forms a region of double stranded RNA. 8.The composition according to claim 3, wherein said nucleic acidcomprises an RNA molecule.
 9. The composition according to claim 8,wherein said RNA molecule is a double stranded RNA molecule.
 10. Thecomposition according to claim 9, wherein said RNA molecule is an siRNA.11. A method of introducing a nucleic acid into a cell, comprisingcontacting a eukaryotic cell with a composition according to claim 3.12. A kit for transfecting a cell, comprising a lipid according toclaim
 1. 13. A composition comprising a lipid according to claim 1 and apeptide or protein.
 14. A method of introducing a peptide or proteininto a cell, comprising contacting a eukaryotic cell with a compositionaccording to claim
 13. 15. A kit for introducing a desired peptide orprotein into a cell, comprising a lipid according to claim
 1. 16. Amethod of introducing a desired molecule into a tissue, comprisingcontacting said tissue with a composition comprising said desiredmolecule and a lipid according to claim
 1. 17. A method according toclaim 16, wherein said desired molecule is a nucleic acid, a peptide, ora protein.